Probe set for analyzing a DNA sample and method for using the same

ABSTRACT

This disclosure provides, inter alia, a probe system probe system for analyzing a nucleic acid sample. In some embodiments, the probe system may comprise: a set of identifier oligonucleotides of sequence B, a set of splint oligonucleotides of formula X′-A′-B′-Z′, wherein sequence A′ is complementary to a genomic fragment and sequence B′ is complementary to at least one member of the set of identifier oligonucleotides, and one or more probe sequences comprising X and Z. Each splint oligonucleotide is capable of hybridizing to the probe sequences, a member of the set of identifier oligonucleotides and a genomic fragment, thereby producing a ligatable complex of formula X-A-B-Z. The probe system can be used to identify a chromosome aneuploidy in cell free DNA, for example.

CROSS-REFERENCING

This application is a continuation of U.S. application Ser. No.16/679,098, filed on Nov. 8, 2019, which is a continuation of U.S.application Ser. No. 15/268,322, filed on Sep. 16, 2016, which claimsthe benefit of provisional application Ser. No. 62/220,746, filed onSep. 18, 2015, which applications are incorporated by reference hereinin their entirety.

BACKGROUND

Cell free DNA (“cfDNA”) can be analyzed to provide a prognosis,diagnosis or a prediction of a response to a treatment for a variety ofdiseases and conditions, including various cancers, transplant failureor success, inflammatory diseases, infectious disease and fetalaneuploidy.

Cell-free fetal DNA (cffDNA) is present in the blood of a pregnantfemale. This discovery led to the possibility of performing non-invasiveprenatal testing (NIPT) of a fetus using a blood sample from thepregnant female. Invasive prenatal tests (e.g., amniocentesis orchorionic villi sampling (CVS)) can be stressful for the mother and somebelieve such procedures may increase the risk of miscarriage. NIPT canprovide information related to a variety of genetic defects, includingDown syndrome (trisomy chromosome 21), Patau syndrome (trisomy 13), andEdwards syndrome (trisomy 18). Such methods should be highly robust as afalse positive may lead to unnecessary medical procedures, and a falsenegative may deprive the expectant mother of understanding the availablemedical options.

There are many technical hurdles associated with implementing anon-invasive prenatal test on a clinical scale. For example, many NIPTefforts have focused on the analysis of cffDNA to identify copy numberchanges in particular sequences (e.g., sequences from chromosome 21).However, such methods are difficult to implement in a robust waybecause, in part, the vast majority of cfDNA in a blood sample ismaternal in origin and in many cases only a very small amount (e.g., onaverage ˜10% and down to about 3%) is from the fetus. For example, thepresence or absence of an extra copy of a chromosome (such as chromosome21) in the fetus may be determined by comparing the copy number ofsequences corresponding to chromosome 21 to the copy number of sequencescorresponding to an autosomal chromosome. While such methods soundattractive, they are in fact challenging because the fractionalconcentration of fetal DNA relative to maternal DNA in maternal bloodcan be as low as 3%. As such, for every 1000 sequences correspondingchromosome 21 that are in the maternal bloodstream, only a smallpercentage of those sequences (e.g., 30 sequences if the fetal fractionis 3%) are from the fetus. Thus, an extra copy of a chromosome in thefetus will only lead to a relatively small increase in the number ofsequences corresponding to that chromosome in the maternal bloodstream.For example, if the fetal fraction is 4, fetal trisomy 21 will only leadto a 1.5% increase in the number of fragments corresponding tochromosome 21 in the maternal bloodstream. As a result of this problem,statistical rigor can only be achieved by counting large numbers ofsequences corresponding to a chromosomal region that is suspected ofhaving a copy number difference (e.g., at least 1,000 and sometimes atleast 5,000 or more sequences) and comparing that number to a similarnumber for another chromosomal region that is not suspected of having acopy number difference. Being able to consistently and accurately countfragments is paramount to the success of many NIPT methods.

Some NIPT methods use polymerase chain reaction (PCR) to amplify theDNA. PCR is widely used, but it suffers from various limitations thatcan negatively affect the accuracy of the results. PCR can introducesequence artifacts and create amplification bias in a sample. PCRsequence artifacts are errors introduced into the DNA sequence of thePCR amplified product by the PCR reaction. PCR sequence artifacts can becaused by various events, such as by the formation of chimeric molecules(e.g., two different pieces of DNA joined end to end), the formation ofheteroduplex DNA (e.g., the hybridization of two different DNA moleculesto each other) and by errors made by the amplification enzyme (e.g., byTaq DNA polymerase placing a mismatched nucleotide onto the DNAtemplate). Sequence bias from PCR is a skewing of the distribution ofPCR products compared to the original sample. PCR sequence bias can becaused by various events, such as intrinsic differences in theamplification efficiency of templates or inhibition of amplification dueto self-annealing of DNA templates. PCR errors result in an unequalamplification of the different DNA molecules so that the amplifiedsample is no longer representative of the original sample. PCR is alsonotoriously sensitive to exogenous DNA contamination from theenvironment. Due to the exponential amplification of DNA during PCR,even very small amounts of exogenous DNA contamination in a PCR reactioncan lead to highly inaccurate results. Exogenous DNA contamination canbe introduced from aerosolized droplets floating in the air or can betransferred into a reaction from contaminated equipment.

Use of rolling-circle amplification (RCA) to analyze cfDNA in maternalblood avoids many of the problems associated with PCR. However, RCAproducts are not very easy to quantify in a way that providesstatistical robustness. At a practical level, although the absolutenumbers of products in an RCA reaction may be sufficiently high toprovide statistical robustness, different RCA products may be amplifiedand detected at different efficiencies and, as such, consistentlydetecting tens or hundreds of thousands of RCA products evenly has beenchallenging.

SUMMARY

Described herein, among other things, is a system of probes foranalyzing a nucleic acid sample. The probes may be designed in such away that they can be ligated to target fragments of genomic DNA (alsoreferred to herein as “target sequences” or merely “fragments”) fromdifferent loci (e.g., different chromosomes) to produce circular DNAmolecules. The circular DNA molecules, even if they contain fragmentsfrom different chromosomes, all contain the same “backbone” sequence.Further, in some embodiments, all of the circular DNA molecules thatcontain a fragment from the same locus contain the same locus-specificidentifier sequence, i.e., a locus-specific barcode. In theseembodiments, the circular DNA molecules can be amplified using a primerthat hybridizes to a sequence in the backbone, and the locus from whichthe cloned fragment is derived can be detected by hybridizing the RCAproducts to a labeled oligonucleotide that hybridizes to thelocus-specific identifier sequence. As would be apparent, thisembodiment of the method can be multiplexed using multiplelocus-specific identifier sequences and distinguishably-labeledoligonucleotides that hybridize to those sequences. Because all of thecircular products have the same backbone and only differ from oneanother by the sequence of the cloned fragment and the locus-specificbarcode, the RCA products amplified from those products amplifiedconsistently, and the locus to which those RCA products correspond canbe detected with accuracy. A method that employs the probe system, aswell as a kit for practicing the same, are also provided.

As will be discussed in greater detail below, in certain cases themethod may be used to detect chromosome abnormalities (e.g., trisomy 21)in a fetus using a sample of cfDNA from a pregnant female carrying thefetus.

A probe system for analyzing a nucleic acid sample is provided. In someembodiments, the probe system may comprise: (a) a set of identifieroligonucleotides of sequence B; (b) a set of splint oligonucleotides offormula X′-A′-B′-Z′, wherein: within the set: (i) sequences A′ and B′vary, and (ii) sequences X′ and Z′ are different from each other and arenot variable; and, within each splint oligonucleotide: (i) sequence A′is complementary to a genomic fragment of the nucleic acid sample and(ii) sequence B′ is complementary to at least one member of the set ofidentifier oligonucleotides; and (c) one or more probe sequencescomprising X and Z, where sequences X and Z are not variable andhybridize to sequences X′ and Z′; where each splint oligonucleotide iscapable of hybridizing to: (i) the probe sequences, (ii) a member of theset of identifier oligonucleotides and, (iii) the genomic fragment,thereby producing a ligatable complex of formula X-A-B-Z. In someembodiments, the different identifier oligonucleotides and theircomplementary sequences B′ identify different chromosomes, e.g.,chromosomes 21, 18 and 13.

In some embodiments, the set of identifier oligonucleotides may compriseat least two (e.g., two, three or four or more) different B sequenceidentifier oligonucleotides and, within the set of splintoligonucleotides, there are at least 100 different A′ sequences and atleast two different B′ sequences that are complementary to at least twodifferent identifier oligonucleotides.

In some embodiments, each identifier oligonucleotide or itscomplementary B′ sequence in a splint oligonucleotide may correspond tothe genomic fragment.

In some embodiments, each identifier oligonucleotide or itscomplementary B′ sequence in a splint oligonucleotide may indicate alocus in a genome from which the genomic fragment is derived.

In some embodiments, each identifier oligonucleotide or itscomplementary B′ sequence in a splint oligonucleotide may indicate thechromosome from which the genomic fragment is derived.

In some embodiments, the genomic fragment is from a mammalian genome.

In some embodiments, each identifier oligonucleotide or itscomplementary B′ sequence in a splint oligonucleotide may identify oneor more of chromosome 21, chromosome 18 and chromosome 13.

In some embodiments, the genomic fragment may be a restriction fragment.

In some embodiments, the one or more probe sequences of (c) may furthercomprise an oligonucleotide comprising sequence Y, and wherein theligatable complex is linear.

In some embodiments, the probe system may further comprise a pair of PCRprimers that hybridize to the one or more probes of (c).

In some embodiments, the one or more probe sequences of (c) may comprisea backbone probe of formula X-Y-Z, where Y comprises an oligonucleotidesequence, such that the ligatable complex is a circular ligatablecomplex of formula X-A-B-Z-Y, where sequence Y joins sequences X and Z.

In some embodiments, the probe system may further comprise a rollingcircle amplification primer that hybridizes to a sequence in thebackbone probe.

In some embodiments, the probe system may further comprise (A) a rollingcircle amplification primer that hybridizes a sequence to the backboneprobe; and (B) up to four distinguishably labeled detectionoligonucleotides, wherein each of the distinguishable labeled detectionoligonucleotides hybridizes to a B′ sequence.

A method of sample analysis is also provided. In some embodiments, themethod may comprise: (a) hybridizing any embodiment of the probe systemsummarized above with a test genomic sample that comprises genomicfragments to produce ligatable complexes of formula X-A-B-Z; (b)ligating the ligatable complexes to produce product DNA molecules offormula X-A-B-Z; and (c) counting the product DNA moleculescorresponding to each locus identifier of sequence B.

In some embodiments, the counting may be done by sequencing product DNAmolecules, or amplification products thereof, to produce sequence reads,and counting the number of sequence reads comprising each sequence of Bor complement thereof.

In some embodiments, the product DNA molecules may be circular, and thecounting may comprise amplifying the product DNA molecules by rollingcircle amplification, and counting the number amplification productscomprising each sequence of B or complement thereof. In theseembodiments, the method may comprise labelling the RCA products usingdistinguishably labeled probes that hybridize to sequence B′, and thecounting is done by counting the number of RCA products for eachdistinguishable label.

In some embodiments, the method may comprise: i. depositing the RCAproducts on a planar support; and ii. counting the number of theindividual labeled RCA products in an area of the support. In theseembodiments, the support may be a glass slide or a porous transparentcapillary membrane, for example.

In some embodiments, the different sequences of B and theircomplementary sequences B′ identify different chromosomes, and themethod further comprises comparing the number of product DNA moleculescomprising a first sequence of either B or B′ to the number of productDNA molecules comprising a second sequence of either B or B′ todetermine if the genomic sample has an aneuploidy.

In some embodiments, the method may comprise comparing the countingresults of step (c) with the counting results obtained from one or morereference samples.

In some embodiments, the test genomic sample may be from a patient thatis suspected or at risk of having a disease or condition, and thecounting results of step (c) provides an indication of whether thepatient, or fetus thereof, has the disease or condition.

In some embodiments, the disease or condition may be a cancer, aninfectious disease, an inflammatory disease, a transplant rejection, ora trisomy.

In some embodiments, the fragments are restriction fragments.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 schematically illustrates some of the features of the presentprobe system.

FIG. 2 schematically illustrates how sequence B serves to identify thelocus of sequence A.

FIG. 3 schematically illustrates some exemplary probe systemconfigurations.

FIG. 4 schematically illustrates some of the features of an embodimentof a subject method.

FIG. 5 schematically illustrates some of the features of oneimplementation of a subject method.

FIG. 6 schematically illustrates the design of probe systems.

FIG. 7 shows data obtained using two different probe systems.

FIG. 8 shows data obtained from the analysis of clinical samples.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Numeric ranges are inclusive of the numbers defining the range. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; and, amino acid sequences are written left to right inamino to carboxy orientation, respectively.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. For example, the term “a primer”refers to one or more primers, i.e., a single primer and multipleprimers. It is further noted that the claims can be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

The term “nucleotide” is intended to include those moieties that containnot only the known purine and pyrimidine bases, but also otherheterocyclic bases that have been modified. Such modifications includemethylated purines or pyrimidines, acylated purines or pyrimidines,alkylated riboses or other heterocycles. In addition, the term“nucleotide” includes those moieties that contain hapten or fluorescentlabels and may contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups, are functionalized as ethers, amines, or the likes.

The term “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length, e.g., greater than about 2bases, greater than about 10 bases, greater than about 100 bases,greater than about 500 bases, greater than 1000 bases, up to about10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotidesor ribonucleotides, and may be produced enzymatically or synthetically(e.g., PNA as described in U.S. Pat. No. 5,948,902 and the referencescited therein) which can hybridize with naturally occurring nucleicacids in a sequence specific manner analogous to that of two naturallyoccurring nucleic acids, e.g., can participate in Watson-Crick basepairing interactions. Naturally-occurring nucleotides include guanine,cytosine, adenine, thymine, uracil (G, C, A, T and U respectively). DNAand RNA have a deoxyribose and ribose sugar backbone, respectively,whereas PNA's backbone is composed of repeating N-(2-aminoethyl)-glycineunits linked by peptide bonds. In PNA various purine and pyrimidinebases are linked to the backbone by methylene carbonyl bonds. A lockednucleic acid (LNA), often referred to as inaccessible RNA, is a modifiedRNA nucleotide. The ribose moiety of an LNA nucleotide is modified withan extra bridge connecting the 2′ oxygen and 4′ carbon. The bridge“locks” the ribose in the 3′-endo (North) conformation, which is oftenfound in the A-form duplexes. LNA nucleotides can be mixed with DNA orRNA residues in the oligonucleotide whenever desired. The term“unstructured nucleic acid”, or “UNA”, is a nucleic acid containingnon-natural nucleotides that bind to each other with reduced stability.For example, an unstructured nucleic acid may contain a G′ residue and aC′ residue, where these residues correspond to non-naturally occurringforms, i.e., analogs, of G and C that base pair with each other withreduced stability, but retain an ability to base pair with naturallyoccurring C and G residues, respectively. Unstructured nucleic acid isdescribed in US20050233340, which is incorporated by reference hereinfor disclosure of UNA.

The term “oligonucleotide” as used herein denotes a single-strandedmultimer of nucleotides of from about 2 to 200 nucleotides, up to 500nucleotides in length. Oligonucleotides may be synthetic or may be madeenzymatically, and, in some embodiments, are 30 to 150 nucleotides inlength. Oligonucleotides may contain ribonucleotide monomers (i.e., maybe oligoribonucleotides) or deoxyribonucleotide monomers. Anoligonucleotide may be 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51to 60,61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200 nucleotides inlength, for example.

The term “primer” as used herein refers to an oligonucleotide that iscapable of acting as a point of initiation of synthesis when placedunder conditions in which synthesis of a primer extension product, whichis complementary to a nucleic acid strand, is induced, i.e., in thepresence of nucleotides and an inducing agent such as a DNA polymeraseand at a suitable temperature and pH. The primer may be single-strandedand must be sufficiently long to prime the synthesis of the desiredextension product in the presence of the inducing agent. The exactlength of the primer will depend upon many factors, includingtemperature, source of primer and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence or fragment, the oligonucleotide primer typically contains15-25 or more nucleotides, although it may contain fewer nucleotides.The primers herein are selected to be substantially complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

The term “hybridization” or “hybridizes” refers to a process in which anucleic acid strand anneals to and forms a stable duplex, either ahomoduplex or a heteroduplex, under normal hybridization conditions witha second complementary nucleic acid strand, and does not form a stableduplex with unrelated nucleic acid molecules under the same normalhybridization conditions. The formation of a duplex is accomplished byannealing two complementary nucleic acid strands in a hybridizationreaction. The hybridization reaction can be made to be highly specificby adjustment of the hybridization conditions (often referred to ashybridization stringency) under which the hybridization reaction takesplace, such that hybridization between two nucleic acid strands will notform a stable duplex, e.g., a duplex that retains a region ofdouble-strandedness under normal stringency conditions, unless the twonucleic acid strands contain a certain number of nucleotides in specificsequences which are substantially or completely complementary. “Normalhybridization or normal stringency conditions” are readily determinedfor any given hybridization reaction. See, for example, Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NewYork, or Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press. As used herein, the term “hybridizing”or “hybridization” refers to any process by which a strand of nucleicacid binds with a complementary strand through base pairing.

A nucleic acid is considered to be “selectively hybridizable” to areference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Moderate and high stringency hybridizationconditions are known (see, e.g., Ausubel, et al., Short Protocols inMolecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold SpringHarbor, N.Y.). One example of high stringency conditions includeshybridization at about 42C in 50% formamide, 5×SSC, 5×Denhardt'ssolution, 0.5% SDS and 100 ug/ml denatured carrier DNA followed bywashing two times in 2×SSC and 0.5% SDS at room temperature and twoadditional times in 0.1×SSC and 0.5% SDS at 42° C.

The term “barcode sequence” or “molecular barcode”, as used herein,refers to a unique sequence of nucleotides used to a) identify and/ortrack the source of a polynucleotide in a reaction and/or b) count howmany times an initial molecule is sequenced (e.g., in cases wheresubstantially every molecule in a sample is tagged with a differentsequence, and then the sample is amplified). A barcode sequence may beat the 5′-end, the 3′-end or in the middle of an oligonucleotide.Barcode sequences may vary widely in size and composition; the followingreferences provide guidance for selecting sets of barcode sequencesappropriate for particular embodiments: Casbon (Nuc. Acids Res. 2011, 22e81), Brenner, U.S. Pat. No. 5,635,400; Brenner et al, Proc. Natl. Acad.Sci., 97: 1665-1670 (2000); Shoemaker et al, Nature Genetics, 14:450-456 (1996); Morris et al, European patent publication 0799897A1;Wallace, U.S. Pat. No. 5,981,179; and the like. In particularembodiments, a barcode sequence may have a length in range of from 4 to36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20nucleotides.

The term “sequencing”, as used herein, refers to a method by which theidentity of at least 10 consecutive nucleotides (e.g., the identity ofat least 20, at least 50, at least 100 or at least 200 or moreconsecutive nucleotides) of a polynucleotide are obtained.

The term “next-generation sequencing” refers to the so-calledparallelized sequencing-by-synthesis or sequencing-by-ligation platformscurrently employed by, e.g., Illumina, Life Technologies, and Roche etc.Next-generation sequencing methods may also include nanopore sequencingmethods or electronic-detection based methods such as, e.g., Ion Torrenttechnology commercialized by Life Technologies.

The term “duplex,” or “duplexed,” as used herein, describes twocomplementary polynucleotides that are base-paired, i.e., hybridizedtogether.

The terms “determining,” “measuring,” “evaluating,” “assessing,”“assaying,” and “analyzing” are used interchangeably herein to refer toforms of measurement, and include determining if an element is presentor not. These terms include both quantitative and/or qualitativedeterminations. Assessing may be relative or absolute.

The term “affinity tag”, as used herein, refers to moiety that can beused to separate a molecule to which the affinity tag is attached fromother molecules that do not contain the affinity tag. An “affinity tag”is a member of a specific binding pair, i.e. two molecules where one ofthe molecules through chemical or physical means specifically binds tothe other molecule. The complementary member of the specific bindingpair, referred to herein as a “capture agent” may be immobilized (e.g.,to a chromatography support, a bead or a planar surface) to produce anaffinity chromatography support that specifically binds the affinitytag. In other words, an “affinity tag” may bind to a “capture agent”,where the affinity tag specifically binds to the capture agent, therebyfacilitating the separation of the molecule to which the affinity tag isattached from other molecules that do not contain the affinity tag.

As used herein, the term “biotin moiety” refers to an affinity agentthat includes biotin or a biotin analogue such as desthiobiotin,oxybiotin, 2′-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin,etc. Biotin moieties bind to streptavidin with an affinity of at least10⁻⁸M. A biotin affinity agent may also include a linker, e.g.,-LC-biotin, -LC-LC-Biotin, -SLC-Biotin or -PEG_(n)-Biotin where n is3-12.

The term “terminal nucleotide”, as used herein, refers to the nucleotideat either the 5′ or the 3′ end of a nucleic acid molecule. The nucleicacid molecule may be in double-stranded form (i.e., duplexed) or insingle-stranded form.

The term “ligating”, as used herein, refers to the enzymaticallycatalyzed joining of the terminal nucleotide at the 5′ end of a firstDNA molecule to the terminal nucleotide at the 3′ end of a second DNAmolecule.

The terms “plurality”, “set” and “population” are used interchangeablyto refer to something that contains at least 2 members. In certaincases, a plurality may have at least 10, at least 100, at least 100, atleast 10,000, at least 100,000, at least 10⁶, at least 10⁷, at least 10⁸or at least 10⁹ or more members.

The term “digesting” is intended to indicate a process by which anucleic acid is cleaved by a restriction enzyme. In order to digest anucleic acid, a restriction enzyme and a nucleic acid containing arecognition site for the restriction enzyme are contacted underconditions suitable for the restriction enzyme to work. Conditionssuitable for activity of commercially available restriction enzymes areknown, and supplied with those enzymes upon purchase.

An “oligonucleotide binding site” refers to a site to which anoligonucleotide hybridizes in a target polynucleotide or fragment. If anoligonucleotide “provides” a binding site for a primer, then the primermay hybridize to that oligonucleotide or its complement.

The term “separating”, as used herein, refers to physical separation oftwo elements (e.g., by size or affinity, etc.) as well as degradation ofone element, leaving the other intact.

The term “reference chromosomal region,” as used herein refers to achromosomal region of known nucleotide sequence, e.g. a chromosomalregion whose sequence is deposited at NCBI's Genbank database or otherdatabases, for example.

The term “strand” as used herein refers to a nucleic acid made up ofnucleotides covalently linked together by covalent bonds, e.g.,phosphodiester bonds.

In a cell, DNA usually exists in a double-stranded form, and as such,has two complementary strands of nucleic acid referred to herein as the“top” and “bottom” strands. In certain cases, complementary strands of achromosomal region may be referred to as “plus” and “minus” strands, the“first” and “second” strands, the “coding” and “noncoding” strands, the“Watson” and “Crick” strands or the “sense” and “antisense” strands. Theassignment of a strand as being a top or bottom strand is arbitrary anddoes not imply any particular orientation, function or structure. Thenucleotide sequences of the first strand of several exemplary mammalianchromosomal regions (e.g., BACs, assemblies, chromosomes, etc.) isknown, and may be found in NCBI's Genbank database, for example.

The term “top strand,” as used herein, refers to either strand of anucleic acid but not both strands of a nucleic acid. When anoligonucleotide or a primer binds or anneals “only to a top strand,” itbinds to only one strand but not the other. The term “bottom strand,” asused herein, refers to the strand that is complementary to the “topstrand.” When an oligonucleotide binds or anneals “only to one strand,”it binds to only one strand, e.g., the first or second strand, but notthe other strand.

The term “covalently linking” refers to the production of a covalentlinkage between two separate molecules, e.g., the top and bottom strandsof a double stranded nucleic acid. Ligating is a type of covalentlinking.

The term “denaturing,” as used herein, refers to the separation of atleast a portion of the base pairs of a nucleic acid duplex by placingthe duplex in suitable denaturing conditions. Denaturing conditions arewell known in the art. In one embodiment, in order to denature a nucleicacid duplex, the duplex may be exposed to a temperature that is abovethe melting temperature of the duplex, thereby releasing one strand ofthe duplex from the other. In certain embodiments, a nucleic acid may bedenatured by exposing it to a temperature of at least 90° C. for asuitable amount of time (e.g., at least 30 seconds, up to 30 mins).Nucleic acids may also be denatured chemically (e.g., using urea orNaOH).

As used herein, the term “label” refers to any atom or molecule that canbe used to provide a detectable (preferably quantifiable) effect, andthat can be attached to a nucleic acid or protein. Labels include butare not limited to dyes and radiolabels such as ³²P; binding moietiessuch as biotin; haptens such as digoxigenin; luminogenic, phosphorescentor fluorogenic moieties; and fluorescent dyes alone or in combinationwith moieties that can suppress or shift emission spectra byfluorescence resonance energy transfer (FRET). Labels may providesignals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, and the like. A label may be a charged moiety (positive ornegative charge) or alternatively, may be charge neutral. Labels caninclude or consist of a nucleic acid or a protein sequence, so long asthe sequence comprising the label is detectable.

The terms “labeled oligonucleotide” and “labeled probe” as used herein,refer to an oligonucleotide that has an affinity tag (e.g., a biotinmoiety), an oligonucleotide modified with atoms or groups enablingseparation or detection (e.g., bromo-deoxyuridine, or colloidal goldparticles conferring different density), and an oligonucleotide modifiedwith or an optically detectable label (e.g., a fluorescence or anothertype of light emitting label). Oligonucleotides that contain onlynaturally occurring nucleotides are not labeled oligonucleotides.

The term “extending”, as used herein, refers to the extension of aprimer by the addition of nucleotides using a polymerase. If a primerthat is annealed to a nucleic acid is extended, the nucleic acid acts asa template for an extension reaction.

As used herein, the term “respective ends”, in the phrase “ligating afirst and second oligonucleotides to the respective ends of a fragment”is intended to mean that one oligonucleotide is added to one end of thefragment and another oligonucleotide is added to the other end of thetarget fragment.

As used herein, the term “ligatably adjacent” in the context of twooligonucleotide sequences that are ligatably adjacent to one another,means that there are no intervening nucleotides between twooligonucleotides and they can be ligated to one another.

As used herein, the term “splint oligonucleotide”, as used herein,refers to an oligonucleotide that, when hybridized to two or more otherpolynucleotides, acts as a “splint” to position the polynucleotides nextto one another so that they can be ligated together, as illustrated inFIG. 1 .

As used herein, the term “a circular nucleic acid molecule” refers to astrand that is in the form of a closed circle that has no free 3′ or 5′ends.

The term “corresponds to” and grammatical equivalents, e.g.,“corresponding”, as used herein refers to a specific relationshipbetween the elements to which the term refers. For example, an RCA thatcorresponds to a sequence in a genome contains the same nucleotidesequence as the sequence in the genome.

Certain polynucleotides described herein may be referred by a formula(e.g., “X′-A′-B′-Z′”). Unless otherwise indicated the polynucleotidesdefined by a formula may be oriented in the 5′ to 3′ direction or the 5′to 3′ direction. For example, polynucleotides defined by the formula“X′-A′-B′-Z′” may be “5′-X′-A′-B′-Z′-3′” or “3′-X′-A′-B′-Z′-5′”. Thecomponents of the formula, e.g., “A”, “X” and “B”, etc., refer toseparately definable sequences of nucleotides within a polynucleotide,where, unless implicit from the context (e.g., in the case of a“ligatable” complex of a particular formula), the sequences are linkedtogether covalently such that a polynucleotide described by a formula isa single molecule. In many cases the components of the formula areimmediately adjacent to one another in the single molecule. Followingconvention, the complement of a sequence shown in a formula will beindicated with a prime (′) such that the complement of sequence “A” willbe “A′”. Moreover, unless otherwise indicated or implicit from thecontext, a polynucleotide defined by a formula may have additionalsequence, a primer binding site, a molecular barcode, a promoter, or aspacer, etc., at its 3′ end, its 5′ end or both the 3′ and 5′ ends. If apolynucleotide defined by a formula is described as being circular thenthe ends of those molecules are joined together, either directly orindirectly. For example, in the case of circular complexes of formulaX-A-B-Z-Y, then the 5′ end of the molecule is joined, directly orindirectly, to 3′ end of the molecule to produce a circle. As would beapparent, the various component sequences of a polynucleotide (e.g., A,B, C, X, Y, Z, etc.) may independently be of any desired length as longas they capable of performing the desired function (e.g., hybridizing toanother sequence). For example, the various component sequences of apolynucleotide may independently have a length in the range of 8-80nucleotides, e.g., 10-50 nucleotides or 12-30 nucleotides.

The term “ligatable complex”, e.g., of formula X-A-B-Z, refers to acomplex in which the various oligonucleotides are ligatably adjacent toone another (in a circular or linear form), held together by a splintoligonucleotide, as shown in FIG. 1 .

The term “ligatable circular complex”, e.g., of formula X-A-B-Z-Y,refers to a circular complex in which the various oligonucleotides areligatably adjacent to one another in a circle, held together by a splintoligonucleotide.

The terms “locus” “genomic locus” as used herein, refer to a definedregion of a genome, e.g., an animal or plant genome such as the genomeof a human, monkey, rat, fish or insect or plant. A locus can be aregion of a chromosome that is as short as a 100 kb, and can be as longas a chromosome arm or an entire chromosome.

The terms “first locus” and “second locus” refer to different loci,i.e., different regions in a genome, e.g., different chromosome arms ordifferent chromosomes.

The terms “fragments of a locus” refers to a population of definedfragments (which may be made using a restriction enzyme or byre-programming an RNA-guided endonuclease such as CAS9) of a particularlocus. Not all fragments of a locus need to be analyzed. Because thesequences of various genomes have been published, design ofoligonucleotides that hybridize to a fragment of a locus is routine.

The term “complementary to a fragment” refers to a sequence that iscomplementary to a strand (either the top or the bottom strand) of afragment.

The term “genomic sequence”, as used herein, refers to a sequence thatoccurs in a genome.

The term “variable”, in the context of two or more nucleic acidsequences that are variable, refers to two or more nucleic acids thathave different sequences of nucleotides relative to one another. Inother words, if the polynucleotides of a population have a variablesequence or a particular sequence “varies”, then the nucleotide sequenceof the polynucleotide molecules of the population varies from moleculeto molecule. The term “variable” is not to be read to require that everymolecule in a population has a different sequence to the other moleculesin a population.

If two nucleic acids (e.g., sequences A and A′) are “complementary”,they hybridize with one another under high stringency conditions. Inmany cases, two sequences that are complementary have at least 10, e.g.,at least 12, at least 15, at least 20 or at least 25 nucleotides ofcomplementarity and in certain cases may have one, two or threenon-complementary bases.

The term “identifies”, in the context of a sequence that identifies alocus, refers to a molecular barcode is unique for the locus. Such asequence is not from the locus itself, but rather it is a molecularbarcode—usually having a sequence that is not present in the samplebeing analyzed—that is added to the fragments of a locus that are beinganalyzed and that identifies those fragments as being from the locus.For example, if fragments from a first locus are ligated to a firstidentifier sequence and fragments from a second locus are ligated to asecond identifier sequence, then the source of those fragments (thelocus to which they correspond) can be determined by detecting whichidentifier sequence has been ligated to those fragments.

The term “inverted orientation” in the context of two sequences thathybridize to other sequences in an inverted orientation, refers to astructure in which the 5′ and 3′ ends of one of the sequences arehybridized to the other in a way in which the ends are facing oneanother, as illustrated at the top of FIG. 3B.

As used herein, the term “rolling circle amplification” or “RCA” forshort refers to an isothermal amplification that generates linearconcatemerized copies of a circular nucleic acid template using astrand-displacing polymerase. RCA is well known in the molecular biologyarts and is described in a variety of publications including, but notlimited to Lizardi et al (Nat. Genet. 1998 19:225-232), Schweitzer et al(Proc. Natl. Acad. Sci. 2000 97:10113-10119), Wiltshire et al (Clin.Chem. 2000 46:1990-1993) and Schweitzer et al (Curr. Opin. Biotech 200112:21-27), which are incorporated by reference herein.

As used herein, the term “rolling circle amplification products” refersto the concatamerized products of a rolling circle amplificationreaction. As used herein, the term “fluorescently labeled rolling circleamplification products” refers to rolling circle amplification productsthat have been fluorescently labeled by, e.g., hybridizing afluorescently labeled oligonucleotide to the rolling circleamplification products or other means (e.g., by incorporating afluorescent nucleotide into the product during amplification).

As used herein, the term “area”, in the context of an area of a supportor an area of an image, refers to a contiguous or non-contiguous area.For example, if a method involves counting the number of labeled RCAproducts in an area, the area in which the RCA products are counted maybe a single, contiguous space or multiple non-contiguous spaces.

As used herein, the term “imaging” refers to a process by which opticalsignals from the surface of an object are detected and stored as data inassociation with a location (i.e., a “pixel”). A digital image of theobject can be reconstructed from this data. An area of a support may beimaged using a single image or one or more images.

As used herein, the term “individual labeled RCA products” refers toindividual RCA molecules that are labeled.

As used herein, the term “counting” refers to determining the number ofindividual objects in a greater collection. “Counting” requiresdetecting separate signals from individual objects in a plurality (not acollective signal from the plurality of objects) and then determininghow many objects there are in the plurality by counting the individualsignals. In the context of the present method, “counting” is done bydetermining the number of individual signals in an array of signals.

As used herein, the term “array” with reference to an array of RCAproducts refers to a collection of single RCA products on a planarsurface, where the RCA products are spatially separated from one anotheron the plane of the surface (to the extent allowed by Poissondistribution if the array is truly random). A “random” array is an arraywherein the elements, e.g., RCA products, are distributed on the surfaceof a substrate at positions that are not predetermined. In some cases,the distribution of RCA products on a random array may be described byPoisson statistics, such that, e.g., the distribution of distancesbetween RCA products of a random array is approximated by a Poissondistribution.

Other definitions of terms may appear throughout the specification.

Description of Exemplary Embodiments

Before the various embodiments are described, it is to be understoodthat the teachings of this disclosure are not limited to the particularembodiments described, and as such can, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present teachings will be limited onlyby the appended claims.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. While the present teachings are described in conjunction withvarious embodiments, it is not intended that the present teachings belimited to such embodiments. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, the someexemplary methods and materials are now described.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can need to be independentlyconfirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

Probe Compositions

Some embodiments of the probe system may comprise: (a) a set ofidentifier oligonucleotides of sequence B; (b) a set of splintoligonucleotides of formula X′-A′-B′-Z′, wherein: within the set: (i)sequences A′ and B′ vary, and (ii) sequences X′ and Z′ are differentfrom each other and are not variable; and, within each splintoligonucleotide: (i) sequence A′ is complementary to a genomic fragmentof the nucleic acid sample and (ii) sequence B′ is complementary to atleast one member of the set of identifier oligonucleotides; and (c) oneor more probe sequences comprising X and Z, where sequences X and Z arenot variable and hybridize to sequences X′ and Z′; where each splintoligonucleotide is capable of hybridizing to: (i) the probe sequences,(ii) a member of the set of identifier oligonucleotides and, (iii) thegenomic fragment, thereby producing a ligatable complex of formulaX-A-B-Z. As will be described in greater detail below, in someembodiments the different identifier oligonucleotides and theircomplementary sequences B′ identify different chromosomes, e.g.,chromosomes 21, 18 and 13.

FIG. 1 shows the ligatable complex of formula X-A-B-Z, which structurecharacterizes the present probe system. As shown in FIG. 1 , in thecomplex sequences X, A, B and Z are ligatably adjacent to one another,held in position by a splint oligonucleotide. As noted in FIG. 1 ,sequence A is a target fragment of a genome (e.g., a strand of arestriction fragment), and sequence B identifies the locus (e.g., aparticular region on a chromosome, a particular chromosome arm or aparticular chromosome, etc.) from which the adjacent sequence A isderived. The relationship between sequences A and B is illustrated inFIG. 2 , which illustrates a simple probe set, hybridized to variousgenomic fragments (A₁ to A₆). As shown in FIG. 2 , the genomic fragmentsin the top three complexes (of sequence A₁, A₂, and A₃) are from a firstlocus (e.g., chromosome 21) and the genomic fragments in the bottomthree complexes (of sequence A₄, A₅, and A₆) are from a second locus(e.g., chromosome 18). The locus from which the genomic fragments in thetop three complexes is derived is identified by a single sequence (B₁),and the locus from which the genomic fragments in the bottom threecomplexes is derived is identified by a different sequence (B₂).Sequence X and Z are the same in all illustrated complexes.

As would be apparent, the set of splint oligonucleotides can be ascomplex as desired and, in some embodiments, sequence A′ may have acomplexity of at least 100, at least 1,000, at least 5,000, at least10,000 or at least 50,000 or more, meaning that the splintoligonucleotides can, collectively, hybridize to at least 100, at least1,000, at least 5,000, at least 10,000 or at least 50,000 or morefragments of genomic DNA. Sequence B′ in the set of splintoligonucleotides may be much less diverse because it simply serves as alocus identifier. As such, in the set of splint oligonucleotides,sequence B′ may have a complexity of at least 2, e.g., 3 or 4, althoughsequence B′ may have a complexity of at least 10, at least 100 or atleast 1000 in some implementations. As would be apparent, becausesequence B′ is complementary to sequence B, the complexity of the set oflocus-specific oligonucleotides may be the same as the complexity ofsequence B′. For example, if there are three identifieroligonucleotides, there may be three different B′ sequences. The numberof splint oligonucleotides in a set may vary greatly, depending on thelength of the locus and the number of target fragments. In someembodiments, each set of splint oligonucleotides may contain at least10, at least 50, at least 100, at least 500, at least 1,000, at least5,000, at least 10,000 or at least 50,000 different splintoligonucleotides.

For example, in some embodiments, a set of splint oligonucleotides maycontain: (i) a first sub-population of splint oligonucleotides thatcontain least 100 A′ sequences, e.g., set of A_(1,X)′, x=1-100+, whichare complementary to different fragments of a first locus (e.g.,fragments of chromosome 21 or , e.g., set of A_(1,X), x=1-100+), whereeach of this sub-population of splint oligonucleotides have the same B′sequence, e.g., B₁′; (ii) a second sub-population of splintoligonucleotides that contain least 100 A′ sequences, e.g., set ofA_(2, X)′, x=1-100+, which are complementary to different fragments of asecond locus (e.g., fragments of chromosome 18 or e.g., set of A_(2 X),x=1-100+), where each of this sub-population of splint oligonucleotideshave the same B′ sequence, e.g., B₂′, that is different from the B′sequence of the first (or any other) subpopulation; (iii) a thirdsub-population of splint oligonucleotides that contain least 100 A′sequences, e.g., set of A_(3,X)′, x=1-100+, which are complementary todifferent fragments of a third locus (e.g., fragments of chromosome 18or , e.g., set of A_(3 X), x=1-100+), where each of this sub-populationof splint oligonucleotides have the same B′ sequence, e.g., B₃′, that isdifferent from the B′ sequence of the any other subpopulation; (iv) anoptional fourth sub-population of splint oligonucleotides that containleast 100 A′ sequences, e.g., set of A_(4 X)′, x=1-100+, which arecomplementary to different fragments of a fourth locus (e.g., fragmentsof another chromosome or, e.g., set of A_(4,X), x=1-100+) where each ofthis sub-population of splint oligonucleotides have a B′ sequence, e.g.,B_(4′), that is different from the B′ sequence of any othersubpopulation.

As illustrated in FIG. 3 , the probe system may be configured in avariety of different ways depending on how it is going to be used. Forexample, as illustrated in FIGS. 3A, C and D, sequences X and Z may bein different molecules and, as a result the ligatable complex is linear.In these embodiments, the one or more probes that contain sequences Xand Y may comprise a first oligonucleotide comprising sequence X and asecond oligonucleotide comprising sequence Y. In these embodiments, thefirst and second oligonucleotides do not need to be tailed, as shown inFIG. 1A. In these embodiments, after ligation, the ligation products canbe amplified using, e.g., talked PCR primers that hybridize to sequencesX and Z. In some embodiments (as shown in FIGS. 3C and D), the firstand/or second oligonucleotides may themselves have a tail to provide aprimer binding site to facilitate amplification and counting. In someembodiments, a tail may contain a molecular indexer (e.g., a randomsequence) that allows the number of original ligation products to becounted after those molecules have been amplified and sequenced. Inalternative embodiments, and as shown in FIG. 3B, the one or more probesthat contain sequences X and Y may be a single backbone probe of formulaX-Y-Z. In these embodiments and as shown, the ligatable complex is acircular ligatable complex of formula X-A-B-Z-Y, where sequence Y joinssequences X and Z. In another embodiment illustrated in FIG. 3E, the oneor more probes that contain sequences X and Z may be part of the splintoligonucleotide itself. In these embodiments, the ligation product maybe a “dumbbell” shaped, as shown in FIG. 3E. In these embodiments, theprobe system may further comprising a pair of PCR primers that hybridizeto the one or more probes that comprise sequences X and Z, therebyallowing the central part of the ligation product (i.e., the part thatcontains sequences A and B) to be amplified. In some embodiments, e.g.,the embodiment shown in FIG. 3B, the probe system may further comprisinga rolling circle amplification primer that hybridizes to a sequence inthe backbone probe, thereby facilitating amplification of those productsby rolling circle amplification. In some embodiments, the probe systemmay comprise a rolling circle amplification primer that hybridizes asequence to the backbone probe, and up to four distinguishably labeledoligonucleotides, wherein each of the distinguishable labeledoligonucleotides hybridizes to the complement of a sequence B′. Thiswill be explained in greater detail below.

As such, some embodiments of the probe system may comprise splintoligonucleotides, a backbone probe, and one or more locus-specificoligonucleotide. The probe system may also comprise one or moreamplification primers, such as a rolling circle amplification primerthat hybridizes a sequence in the backbone probe or a pair of PCRprimers that hybridize to sites in the backbone probe, and, optionally,one or more labeled probes that hybridize to the complement of thelocus-specific oligonucleotide.

As noted above, sequence A′ varies between the different members of theset, and the sequences of A′ are each designed to be complementary to adifferent target fragment of a genome. The sequences of A′ mayindependently vary in length and sequence and, in some case, may be inthe range of 8 to 80 nucleotides, e.g., 10 to 60 nucleotides, in length,depending on the length and sequence of the target fragments. SequenceB′ identifies the locus from which the adjacent fragment is derived(e.g., a particular chromosome such as chromosome 18 or 21, etc.).Sequence B′ may be of any suitable length, but in some embodiments it isin the range of 8 to 30 nucleotides in length. Within any single assay,sequences X′ and Z′ are different to one another, and are not variable.Sequence X′ and Z′ may be of any suitable length, but in someembodiments they are independently in the range of 8 to 30 nucleotidesin length, although longer or shorter sequences can be used. The overalllength of the splint oligonucleotides may be in the range of 50 to 200nucleotides. In some embodiments, the splint oligonucleotides may bebiotinylated, thereby allowing ligation products (discussed below) to beisolated from other, unligated, products prior to amplification. Aswould be apparent, sequences X and Z (which may be of any suitablelength but in some embodiments they are independently in the range of 8to 30 nucleotides in length) are not variable and hybridize to sequencesX′ and Z′. The locus-specific oligonucleotide is of sequence B which,again, may be of any suitable length, e.g., in the range of 8 to 30nucleotides in length.

As noted above, the complexes produced using the above-described probesystem may be linear or circular (as shown in FIG. 3 ). FIG. 4illustrates some of the features of the circular embodiment illustratedin FIG. 3B.

As shown in FIG. 4 , in some embodiments, the probe system may comprisethe set of splint oligonucleotides 2 (of formula X′-A′-B′-Z′, which maybe in the 5′ to 3′ or 3′ to 5′ orientation), a backbone probe 6 offormula X-Y-Z, where sequences X and Z are not variable and hybridize tosequences X′ and Z′ in an inverted orientation (i.e., so that the endsof the backbone are pointing toward one another, as shown), and a set oflocus-specific oligonucleotides 8 is of sequence B. Sequence Y in thebackbone probe may be any convenient length, e.g., 20 to100 nucleotides.The overall length of backbone probe 6 may be in the range of 50 to 300nucleotides in length, or longer in certain cases.

As shown in FIG. 4 , the probe set is characterized in the variousoligonucleotides can be hybridized with genomic fragments to produce afirst set of ligatable circular complexes 10 (i.e., a complex in whichthe ends of the backbone probe 6, a locus-specific oligonucleotide 8 anda genomic fragment 4 are ligatably adjacent to one another and heldligatably adjacent to one another by a splint oligonucleotide 2). Asshown in the illustrated example, the backbone probe 6, thelocus-specific oligonucleotide 8 and the fragment 4 hybridize to thefirst splint oligonucleotides 2 to produce a set of ligatable circularcomplexes 10 of formula X-A-B-Z-Y, where sequence Y joins sequences Xand Z. The fragments 4 that are present in this set of ligatablecircular complexes 10 may be from at least 2, at least 5, at least 10,or at least 50 or more different loci (e.g., different chromosomes), andthe identity of the locus from which an adjacent fragment is derived(e.g., the particular chromosomes) is provided by locus-specificoligonucleotide 8, which is the same sequence for each locus. In thisexample sequence A and A′ (which correspond to the sequences ofdifferent genomic fragments) vary, B and B′ (the locus identifier) vary,and sequences X, Y and Z do not vary.

As will be described in greater detail below, in this embodiment, theprobe system (which comprises a first set of splint oligonucleotides 2,a backbone probe 6 and a locus-specific oligonucleotide 8) may behybridized with a sample that comprises fragments of a genome 4 toproduce a first set of ligatable circular complexes of formula X-A-B-Z-Y10, as shown. After ligation of the ligatable circular complexes toproduce a first set of circular DNA molecules 12 of formula X-A-B-Z-Y,the first set of circular DNA molecules can be amplified by rollingcircle amplification (RCA) to produce a first set of RCA products 16.RCA may done using rolling circle amplification primer 14 thathybridizes a sequence in backbone probe 6, as illustrated in FIG. 4 , orPCR primers that hybridize to sites that flank the ligated fragment. Assuch, in certain embodiments, the probe system may additionally comprisea rolling circle amplification primer 14, which primer hybridizes asequence in backbone probe 6, or a pair of PCR primers that hybridize tosites that flank the ligated fragment. After RCA, the “source” of clonedfragment in a particular RCA product 16 (i.e., the locus, e.g., theparticular chromosome, from which the cloned genomic fragment isderived) can then be determined by hybridizing a first labeledoligonucleotide 18 to the complement of sequence B (i.e., B′), or bysequencing. As would be apparent, labeled oligonucleotide 18 maycomprise at least some of sequence B. As such, in certain embodiments,the probe system may additionally comprise a labeled oligonucleotidethat hybridizes to the complement of first locus-specificoligonucleotide 8.

As would be apparent, if sequences from two or more different loci areto be detected in the same reaction, the probe system may compriseadditional, distinguishably labeled oligonucleotides, one for each locusidentifier B, so that both sets of RCA products can be identified at thesame time. In these embodiments, the probe system may further compriseup to four distinguishably labeled oligonucleotides (e.g., B₁, B₂, B₃,B₄), where each of the distinguishable labeled oligonucleotideshybridizes to the complement of a sequence B′ (e.g., B₁′, B₂′, B₃′,B₄′).

As would be apparent, the fragments to which the splint oligonucleotideshybridize are restriction fragments of the genome being analyzed.Further, any of the probes, oligonucleotides, or primers described above(e.g., the backbone probe) may contain a molecular barcode (e.g., anindexing sequence such as a random or semi-random sequence) such thateach circular DNA molecule can be distinguished by the combination ofthe cloned fragment and the barcode, thereby allowing one to count howmany initial molecules were sequenced, even after the molecules havebeen amplified (see, e.g., Casbon et al).

Methods

Also provide herein is a method comprising: (a) hybridizing a probesystem as described above, with a test genomic sample that comprisesfragments of a genome to produce ligatable complexes of formula X-A-B-Z;(b) ligating the ligatable complexes to produce product DNA molecules offormula X-A-B-Z; and (c) counting the product DNA moleculescorresponding to each locus identifier of sequence B. In someembodiments, the counting may be done by sequencing the product DNAmolecules, or amplification products thereof, to produce sequence reads,and counting the number of sequence reads comprising each sequence of B.

In embodiments in which the product DNA molecules are circular, thecounting may comprise amplifying the product DNA molecules by rollingcircle amplification, and counting the number amplification productscomprising each sequence of B. In these embodiments, the method maycomprise labelling the RCA products using distinguishably labeled probesthat hybridize to sequence B, and the counting is done by counting thenumber of RCA products for each distinguishable label. The generalprinciples of one implementation of this method are shown in FIG. 4 . Aswould be apparent, the fragments to which the splint oligonucleotideshybridize can be (independently) top or bottom strands restrictionfragments of the genome being analyzed. These fragments can be generatedby digesting the genome with one or more restriction enzymes (e.g., acombination of enzymes that have a four base recognition sequence), andthen denaturing the digested sample. As such, the fragments being clonedhave defined ends, thereby allowing the design of splintoligonucleotides to clone those fragments. There are other ways togenerate fragments that have defined ends (e.g., methods that use flapendonuclease, exonuclease, gap-fill, etc).

As indicated above, this method may be multiplexed to provide a way toanalyze two or more different loci, as shown in FIG. 5 . With referenceto FIG. 5 , a sample containing fragments of genomic DNA 40 may be: a)hybridized with a probe system 42 comprising: (i) a first set of splintprobes, as described above; (ii) a first-locus specific oligonucleotide,as described above; (iii) a second set of splint probes, as describedabove; (iv) a second locus-specific oligonucleotide, as described above;and, (v) a backbone probe, as described above, to produce a mixture 44comprising first set of ligatable circular complexes of formulaX-A-B-Z-Y (which contain fragments from the first locus, e.g., a firstchromosome, as well as fragments from a second locus, e.g., a secondchromosome). Next, the method comprises (b) ligating the ligatablecircular complexes to produce a mixture of circular DNA molecules 46(which contains the first and second sets of circular DNA molecules),and, after treating the sample with an exonuclease to remove linearnucleic acid molecules, (c) amplifying the circular DNA molecules 46 byrolling circle amplification using a single primer that hybridizes tothe backbone probe, to produce RCA products 48. The locus from whicheach of the fragments contained within each RCA product can then beidentified by hybridizing the RCA products to distinguishably labeledfirst and second oligonucleotide probes, which hybridize to thecomplement of the locus-specific oligonucleotide that is present in eachof the products, to produce a labeled sample 50. In these embodiments,the method may comprise: (d) separately detecting: (i) RCA products thatcontain fragments from a first locus using a labeled probe thathybridizes to a first locus identifier sequence and (ii) RCA productsthat contain fragments from a second locus using a labeled probe thathybridizes to a second locus identifier sequence, wherein the labeledprobes are distinguishably labeled. As noted above, after ligation, ifthe splint oligonucleotides are biotinylated the circular products maybe isolated from unligated products using, e.g., streptavidin beads. Ineither event, the ligated sample may be treated with an exonuclease,thereby removing linear DNA molecules from the reaction. This principlemay be expanded to count to the number of ligation products produced forany number of loci (e.g., 3, 4, up to 10 or up to 100 or more loci).

In some embodiments, the detecting step may (d) comprise: (i) depositingthe RCA products on a support; and, (ii) separately counting the numberof the individual labeled RCA products that are labeled with one labeland the number of individual labeled RCA products labeled with anotherlabel in an area of the support. As would be understood, hybridizationof the labeled oligonucleotides may be done before the RCA products aredistributed on the support, or after the RCA products are distributed onthe support.

In other words, the number of rolling circle amplification productscorresponding to each locus can be estimated by, e.g., distributing theRCA products on the surface of a support (a slide or porous membrane),hybridizing the RCA products using labelled oligonucleotides (e.g.,fluorescently labelled oligonucleotides) and then counting the number ofdiscrete signals in an area of the support, e.g., using a fluorescencereader. The labelling can be done before or after the products have beendistributed on the support and, because each RCA product containsthousands of copies of the same sequences, there should be thousands ofbinding sites for the labelled oligonucleotides, thereby increasing thesignal. In multiplex embodiments (e.g., in which RCA productscorresponding to two different locus are being counted), the RCAproducts corresponding to one locus can be labelled with one fluorophoreand the RCA products corresponding to another locus can be labelled witha different fluorophore, thereby allowing the different RCA products tobe separately counted.

In certain embodiments, the method may comprise (a) filtering a liquidsample containing the rolling circle amplification (RCA) productsthrough a porous transparent capillary membrane, thereby concentratingthe RCA products and producing an array of the RCA products on themembrane; (b) fluorescently labeling the RCA products prior to or afterstep (a); and, (c) counting the number of the individual labeled RCAproducts in an area of the membrane, thereby providing an estimate ofthe number of the labeled RCA products in the sample. In someembodiments, the porous transparent capillary membrane may be a porousanodic aluminum oxide membrane. In these embodiments, the labeling step(b) may done by hybridizing fluorescently labeled oligonucleotides tothe RCA products, prior to or after step (a). In certain embodiments,the method may comprise imaging an area of the membrane to produce oneor more images and counting the number of the individual labeled RCAproducts in the one or more images. Examples of such methods aredescribed in PCT/IB2016/052495, filed on May 2, 2016, which isincorporated by reference herein.

Quantifying signals from individual RCA products is significant because,in many applications (e.g., non-invasive pre-natal diagnosis by analysisof cfDNA), the number of fragments corresponding to particularchromosomes (e.g., chromosome 21) needs to be determined quireaccurately and without bias. Typical analysis methods use PCR which, asis well known, is a very biased procedure in that some sequences areamplified much higher efficiencies than others. This makes PCR-basedstrategies impractical for many diagnostic efforts.

In particular embodiments, the sample may contain multiple populationsof RCA products (e.g., two, three or four or more populations of RCAproducts such as a first population of labeled RCA products and a secondpopulation of RCA products), where the different populations of RCAproducts are distinguishably labeled, meaning that the individualmembers of each of the populations of RCA products labels can beindependently detected and counted, even when the populations are mixed.Suitable distinguishable fluorescent label pairs useful in the subjectmethods include , e.g., Cy-3 and Cy-5 (Amersham Inc., Piscataway, N.J.),Quasar 570 and Quasar 670 (Biosearch Technology, Novato Calif.),Alexafluor555 and Alexafluor647 (Molecular Probes, Eugene, Oreg.),BODIPY V-1002 and BODIPY V1005 (Molecular Probes, Eugene, Oreg.), POPO-3and TOTO-3 (Molecular Probes, Eugene, Oreg.), and POPRO3 TOPRO3(Molecular Probes, Eugene, Oreg.). Further suitable distinguishabledetectable labels may be found in, e.g., Kricka et al. (Ann ClinBiochem. 39:114-29, 2002). For example, the RCA products may be labeledwith any combination of ATTO, ALEXA, CY, or dimeric cyanine dyes such asYOYO, TOTO etc. Other labels may also be used.

In some cases, a population of RCA products can be distinguishablylabeled by labeling it with multiple labels, thereby increasing thepossibilities of multiplexing. For example, in some cases a populationmay be labeled with two distinguishable dyes (e.g., Cy3 and Cy5), which,when read, will be distinguishable from populations that are labeledwith the individual dyes (e.g., Cy3 or Cy5). In some embodiments, afirst population of RCA products represent a “test” population oflabeled RCA products and a second population of RCA products represent a“reference” population of RCA products to which the number of the firstRCA products can be compared. For example, in some embodiments, a firstpopulation of RCA products may correspond to a first chromosomal region(e.g., a first chromosome such as chromosome 21) and a second populationof RCA products may correspond to a second chromosomal region (e.g., asecond chromosome such as chromosome 13 or 18 or a different region ofthe first chromosome) and the number of the first population of RCAproducts and the second population of RCA products can be counted andcompared to determine if there is a difference in the copy number of theregions (indicating that there is duplication or deletion of the testregion). In some embodiments, the sample contains at least a firstpopulation of RCA products and a second population of RCA products,wherein the first and second populations of labeled RCA products aredistinguishably labeled in the labeling step (step (b)). In theseembodiments, the method comprises counting the number of first labeledRCA products in an area of the membrane and counting the number ofsecond labeled RCA products in an area (the same area or a differentarea) of the membrane, thereby providing an estimate of the number offirst and second populations of RCA products in the sample. Thisembodiment may further involve comparing the number of first RCAproducts in the sample to the number of second RCA products in thesample.

In some of these embodiments of the method, the method may compriseimaging the first and second populations of labeled RCA products toproduce one or more images (e.g., a first image and a second image,respectively) and, optionally, (i) counting the number of labeled RCAproducts in the one or more images, thereby providing an estimate of thenumber of first and second populations of labeled RCA products in thesample. The first and second populations of labeled RCA products can beseparately detected using known methods (e.g., using appropriate filtersetc.). These embodiments of the method may further comprise comparingthe number of first labeled RCA products in the sample to the number ofsecond labeled RCA products in the sample. This step of the method mayinvolve counting at least 1,000 (e.g., at least 5,000, at least 10,000,at least 20,000, at least 50,000, at least 100,000, at least 500,000 upto 1M or more) labeled RCA products in the first population at least1,000 (e.g., at least 5,000, at least 10,000, at least 20,000 or atleast 50,000, at least 100,000, at least 500,000 up to 1M or more)labeled RCA products in an area of the membrane and counting, therebyensuring that a difference in copy number can be called with statisticalrigor.

In alternative embodiments, cloned fragments in the DNA molecules (and,optionally, any indexing sequence in the circular DNA molecules) may beamplified by PCR using PCR primers that hybridize to or are the same assites that flank those sequences. In this embodiment, a PCR product canbe amplified using the primers. In this embodiment, the amount of theproduct can be quantifying by any suitable qPCR assay, e.g., a TaqManassay or the like. In another embodiment, the product may be sequenced(with or without amplification). In these embodiments, the amount ofcircular molecules corresponding to each locus can be estimated bycounting the number of sequence reads corresponding to the locus (e.g.,counting how many sequence reads have a particular locus-specificbarcode sequence). In some embodiments, if an indexing sequence is used,the number of circular molecules corresponding to each locus can becounted by determining how many different molecular barcode sequencesare associated with each locus-specific barcode sequence

As would be apparent, in this embodiment, the primers used may containsequences that are compatible with use in, e.g., Illumina's reversibleterminator method, Roche's pyrosequencing method (454), LifeTechnologies' sequencing by ligation (the SOLiD platform) or LifeTechnologies' Ion Torrent platform. Examples of such methods aredescribed in the following references: Margulies et al (Nature 2005 437:376-80); Ronaghi et al (Analytical Biochemistry 1996 242: 84-9);Shendure (Science 2005 309: 1728); Imelfort et al (Brief Bioinform. 200910:609-18); Fox et al (Methods Mol Biol. 2009;553:79-108); Appleby et al(Methods Mol Biol. 2009;513:19-39) and Morozova (Genomics. 200892:255-64), which are incorporated by reference for the generaldescriptions of the methods and the particular steps of the methods,including all starting products, reagents, and final products for eachof the steps.

The test genomic sample may be from a patient that is suspected or atrisk of having a disease or condition, and the results of step (c) anindication of whether the patient, or fetus thereof, has the disease orcondition. In some embodiments, the disease or condition may be acancer, an infectious disease, an inflammatory disease, a transplantrejection, or a chromosomal defect such as a trisomy.

As noted above, in some cases the sample being analyzed using thismethod may be a sample of cfDNA obtained from blood, e.g., from theblood of a pregnant female. In these embodiments, the method may be usedto detect chromosome abnormalities in the developing fetus (as describedabove) or to calculate the fraction of fetal DNA in the sample, forexample.

Illustrative copy number abnormalities that can be detected using themethod include, but are not limited to, trisomy 21, trisomy 13, trisomy18, trisomy 16, XXY, XYY, XXX, monosomy X, monosomy 21, monosomy 22,monosomy 16, and monosomy 15. Further copy number abnormalities that canbe detected using the present method are listed in the following table.

Chromosome Abnormality and Disease Association X: XO (Turner's Syndrome)Y: XXY (Klinefelter Syndrome) Y: XYY (Double Y Syndrome) Y: XXX (TrisomyX Syndrome) Y: XXXX (Four X Syndrome) Y: Xp21 deletion(Duchenne's/Becker Syndrome, congenital adrenal hypoplasia, chronicgranulomatus disease) Y: Xp22 deletion (steroid sulfatase deficiency) Y:Xq26 deletion (X-linked lymphoproliferative disease)  1: 1p somatic(neuroblastoma)  1: monosomy (neuroblastoma)  1: trisomy (neuroblastoma) 2: monosomy (growth retardation, developmental and mental delay, andminor physical abnormalities)  2: trisomy 2q (growth retardation,developmental and mental delay, and minor physical abnormalities)  3:monosomy (Non-Hodgkin's lymphoma)  3: trisomy somatic (Non-Hodgkin'slymphoma)  4: monosomy (Acute non lymphocytic leukemia (ANLL))  4:trisomy somatic (Acute non lymphocytic leukemia (ANLL))  5: 5p (Cri duchat; Lejeune syndrome)  5: 5q somatic (myelodysplastic syndrome)  5:monosomy (myelodysplastic syndrome)  5: trisomy (myelodysplasticsyndrome)  6: monosomy (clear-cell sarcoma)  6: trisomy somatic(clear-cell sarcoma)  7: 7q11.23 deletion (William's syndrome)  7:monosomy (monosomy 7 syndrome of childhood; somatic: renal corticaladenomas; myelodysplastic syndrome)  7: trisomy (monosomy 7 syndrome ofchildhood; somatic: renal cortical adenomas; myelodysplastic syndrome) 8: 8q24.1 deletion (Langer-Giedon syndrome)  8: monosomy(myelodysplastic syndrome; Warkany syndrome; somatic: chronicmyelogenous leukemia)  8: trisomy (myelodysplastic syndrome; Warkanysyndrome; somatic: chronic myelogenous leukemia)  9: monosomy 9p (Alfi'ssyndrome)  9: monosomy 9p (Rethore syndrome)  9: partial trisomy(Rethore syndrome)  9: trisomy (complete trisomy 9 syndrome; mosaictrisomy 9 syndrome) 10: monosomy (ALL or ANLL) 10: trisomy somatic (ALLor ANLL) 11: 11p- (Aniridia; Wilms tumor) 11: 11q- (Jacobsen Syndrome)11: monosomy (myeloid lineages affected (ANLL, MDS)) 11: trisomy somatic(myeloid lineages affected (ANLL, MDS)) 12: monosomy (CLL, Juvenilegranulosa cell tumor (JGCT)) 12: trisomy somatic (CLL, Juvenilegranulosa cell tumor (JGCT)) 13: 13q- (13q-syndrome; Orbeli syndrome)13: 13q14 deletion (retinoblastoma) 13: monosomy (Patau's syndrome) 13:trisomy (Patau's syndrome) 14: monosomy (myeloid disorders (MDS, ANLL,atypical CML) 14: trisomy somatic (myeloid disorders (MDS, ANLL,atypical CML) 15: 15q11-q13 deletion (Prader-Willi, Angelman's syndrome)15: monosomy (Prader-Willi, Angelman's syndrome) 15: trisomy somatic(myeloid and lymphoid lineages affected, e.g., MDS, ANLL, ALL, CLL) 16:16q13.3 deletion (Rubenstein-Taybi) 16: monosomy (papillary renal cellcarcinomas (malignant)) 16: trisomy somatic (papillary renal cellcarcinomas (malignant)) 17: 17p- somatic (17p syndrome in myeloidmalignancies) 17: 17q11.2 deletion (Smith-Magenis) 17: 17q13.3(Miller-Dieker) 17: monosomy (renal cortical adenomas) 17: trisomysomatic (renal cortical adenomas) 17: 17p11.2-12 (Charcot-Marie ToothSyndrome type 1; HNPP) 17: trisomy (Charcot-Marie Tooth Syndrome type 1;HNPP) 18: 18p- (18p partial monosomy syndrome or Grouchy Lamy Thieffrysyndrome) 18: 18q- (Grouchy Lamy Salmon Landry Syndrome) 18: monosomy(Edwards syndrome) 18: trisomy (Edwards syndrome) 19: monosomy (Edwardssyndrome) 19: trisomy (Edwards syndrome) 20: 20p- (trisomy 20p syndrome)20: 20p11.2-12 deletion (Alagille) 20: 20q- (somatic: MDS, ANLL,polycythemia vera, chronic neutrophilic leukemia) 20: monosomy(papillary renal cell carcinomas (malignant)) 20: trisomy somatic(papillary renal cell carcinomas (malignant)) 21: monosomy (Down'ssyndrome) 21: trisomy (Down's syndrome) 22: 22q11.2 deletion (DiGeorge'ssyndrome, velocardiofacial syndrome, conotruncal anomaly face syndrome,autosomal dominant Opitz G/BBB syndrome, Caylor cardiofacial syndrome)22: monosomy (complete trisomy 22 syndrome) 22: trisomy (completetrisomy 22 syndrome)

The method described herein can be employed to analyze genomic DNA fromvirtually any organism, including, but not limited to, plants, animals(e.g., reptiles, mammals, insects, worms, fish, etc.), tissue samples,bacteria, fungi (e.g., yeast), phage, viruses, cadaveric tissue,archaeological/ancient samples, etc. In certain embodiments, the genomicDNA used in the method may be derived from a mammal, where in certainembodiments the mammal is a human. In exemplary embodiments, the genomicsample may contain genomic DNA from a mammalian cell, such as, a human,mouse, rat, or monkey cell. The sample may be made from cultured cellsor cells of a clinical sample, e.g., a tissue biopsy, scrape or lavageor cells of a forensic sample (i.e., cells of a sample collected at acrime scene). In particular embodiments, the nucleic acid sample may beobtained from a biological sample such as cells, tissues, bodily fluids,and stool. Bodily fluids of interest include but are not limited to,blood, serum, plasma, saliva, mucous, phlegm, cerebral spinal fluid,pleural fluid, tears, lactal duct fluid, lymph, sputum, cerebrospinalfluid, synovial fluid, urine, amniotic fluid, and semen. In particularembodiments, a sample may be obtained from a subject, e.g., a human. Insome embodiments, the sample analyzed may be a sample of cfDNA obtainedfrom blood, e.g., from the blood of a pregnant female.

For example, in some embodiments, a sample of DNA may be obtained andthe sample digested with one or more restriction enzymes (or aRNA-guided endonuclease such as cas9) to produce predictable fragments(the median size of which may be in the range of 20-100 bases). Themethod described above may be performed on the digested DNA, and thenumber of fragments corresponding one locus (e.g., one chromosome) canbe compared to the number of fragments corresponding to another locus(e.g., another chromosome) using the method described herein. As noted,the method may be used to identify copy number differences, e.g.,chromosome aneuploidies, that are associated with a disease orcondition.

As noted above, in some cases the sample analyzed may be a sample ofcfDNA obtained from blood, e.g., from the blood of a pregnant female. Inthese embodiments, the method may be used to detect chromosomeabnormalities in the developing fetus or to calculate the fraction offetal DNA in the sample, for example.

Kits

Also provided by this disclosure are kits for practicing the subjectmethods, as described above. In certain embodiments, the kit maycomprise: (a) a set of splint oligonucleotides of formula X′-A′-B′-Z′,wherein: within the set: (i) the sequence of A′ and B′ vary, and (ii)the sequences of X′ and Z′ are different to each other and are notvariable; and within each molecule: (i) sequence A′ is complementary toa fragment of a genome and (ii) sequence B′ identifies the locus fromwhich the genomic fragment that hybridizes to the adjacent A′ sequenceis derived; (b) one or more probes comprising sequences X and Z,wherein: i. sequences X and Z are not variable and hybridize to sequenceX′ and Z′; and (c) a set of locus-specific oligonucleotides of sequenceB; and wherein: each splint oligonucleotide of (a) is capable ofhybridizing to (i) the probe sequences of (b); (ii) a locus-specificoligonucleotide of (c); and, (iii) a genomic fragment of (a), to producea ligatable complex of formula X-A-B-Z, in which sequence B identifiesthe locus of adjacent sequence A. In some embodiments, the one or moreprobes of (b) comprise a first oligonucleotide comprising sequence X anda second oligonucleotide comprising sequence Y. In some embodiments, thekit may further comprise a pair of PCR primers that hybridize to the oneor more probes comprising sequences X and Y. In certain embodiments, theone or more probes of (b) is a backbone probe of formula X-Y-Z, and theligatable complex is a circular ligatable complex of formula X-A-B-Z-Y,where sequence Y joins sequences X and Z, and sequence B identifies thelocus of adjacent sequence A In these embodiments, the kit may furthercomprise a rolling circle amplification primer that hybridizes to asequence in the backbone probe. In these embodiments, the kit maycomprise a plurality of distinguishably labeled oligonucleotides,wherein each of the distinguishable labeled oligonucleotides hybridizesto the complement of a B′ sequence. The kit may additionally contain aligase and/or a strand-displacing polymerase for performing rollingcircle amplification.

The various components of the kit may be present in separate containersor certain compatible components (e.g., the first and second sets ofsplint probes and the first and second locus-specific probes) may beprecombined into a single container, as desired.

In addition to the above-mentioned components, the subject kit mayfurther include instructions for using the components of the kit topractice the subject method.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with additional disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed.

Example I Initial Data Validating Method

The purpose of this experiment is to compare the methods that usebackbone oligonucleotides that are chromosome-specific (e.g., thebackbone oligonucleotide used to capture fragments from a firstchromosome, e.g., chromosome 21, is different from the backboneoligonucleotide used to capture fragments from a second chromosome,e.g., chromosome 18, as described in described in WO2015083001 andWO2015083002), with methods in which the same backbone oligonucleotideis used for all chromosomes examined. This is illustrated in FIG. 6 . Asshown, in the “new” design, the source of a cloned fragment isdetermined using a chromosome-specific sequence (e.g., A or B) that iscloned into the same circular product as the target fragment. In the newmethod, a single backbone oligonucleotide is used (compared to multiplebackbone oligonucleotides in the prior method), and the cloned fragmentsfrom all chromosomes can be amplified using the same RCA primer or asingle pair of PCR primers.

Cell-line DNA (10 ng) was digested denatured and hybridized to the “old”and “new” probe designs. Following hybridization and ligation, theligation reactions were subjected to exonuclease treatment to remove anynon-circularized DNA in the solution. The remaining circular productsserved as templates in an RCA reaction, which produced concatemericcopies of the circular products. These RCA products were labeled withfluorescently labeled oligonucleotides complementary to the “splint”sequence, and deposited to a solid support for detection.

Thirteen cfDNA samples from pregnant women were subjected to the samereaction as described above.

For all reactions, the number of individual objects (RCA products)wascounted in each color. The ratio of the number of objects in color A/Bwas calculated for each sample and the coefficient of variation wascalculated as a measure of precision of the assay. Low coefficient ofvariation enables precise measurements of samples with low fetalfraction. This was illustrated by adding samples containing a lowspike-in amount of trisomy 21 cell-line sample.

According to the data shown in FIG. 7 , the new design generates a lowerCV for both cell-line DNA and cfDNA, enabling a more accuratemeasurement of fetal DNA with chromosomal abnormalities.

Without wishing to be bound to any particular theory, it is believedthat this method may be less sensitive for impurities in the sample.

Example II Analysis of Clinical Samples

cfDNA samples from 26 normal pregnant individuals and 4 individualscarrying a fetus with trisomy 21 was prepared. Blood (10 ml) from eachpatent were centrifuged to separate plasma from red blood cells andbuffy coat. The corresponding plasma (˜3-5 ml/patient) was subjected toa bead-based DNA extraction protocol, resulting in extracted cfDNAdiluted in 50 ul of buffer.

The cfDNA was then subjected to the method herein described above andanalyzed by digital counting of rolling-circle products usingfluorescence microscope. All 4 positive cases were detected above az-score above 3. The CV of the normal samples was calculated to 0.49%demonstrating the high precision of the assay.

The invention claimed is:
 1. A probe system for analyzing a nucleic acidsample, comprising: (a) a set of identifier oligonucleotides of sequenceB; (b) a set of splint oligonucleotides of formula 5′-X′-A′-B′-Z′-3′ or5′-X′-B′-A′-Z′-3′, wherein: within the set: (i) sequences A′ and B′vary, and (ii) sequences X′ and Z′ are different from each other and arenot variable; and, within each splint oligonucleotide: (i) sequence A′is complementary to a genomic fragment of the nucleic acid sample,wherein the genomic fragment is of sequence A, and (ii) sequence B′ iscomplementary to at least one member of the set of identifieroligonucleotides; and (c) one or more oligonucleotides comprisingsequence X and sequence Z, where sequences X and Z are not variable andhybridize to sequences X′ and Z′, respectively; wherein sequence X,sequence Z, a member of the set of identifier oligonucleotides ofsequence B and the genomic fragment of sequence A hybridize to differentsites in the splint oligonucleotide to produce a ligatable complex offormula X-A-B-Z or X-B-A-Z.
 2. The probe system of claim 1, wherein theset of identifier oligonucleotides comprises at least two different Bsequence identifier oligonucleotides and, within the set of splintoligonucleotides, there are: at least 100 different A′ sequences; and,at least two different B′ sequences that are complementary to at leasttwo different identifier oligonucleotides.
 3. The probe system of claim1, wherein each identifier oligonucleotide or its complementary B′sequence in a splint oligonucleotide corresponds to the genomicfragment.
 4. The probe system of claim 1, wherein each identifieroligonucleotide or its complementary B′ sequence in a splintoligonucleotide indicates a locus in a genome from which the genomicfragment is derived.
 5. The probe system of claim 1, wherein eachidentifier oligonucleotide or its complementary B′ sequence in a splintoligonucleotide indicates the chromosome from which the genomic fragmentis derived.
 6. The probe system of claim 1, wherein the genomic fragmentis from a mammalian genome.
 7. The probe system of claim 1, wherein eachidentifier oligonucleotide or its complementary B′ sequence in a splintoligonucleotide identifies one or more of chromosome 21, chromosome 18and chromosome
 13. 8. The probe system of claim 1, wherein the genomicfragment is a restriction fragment.
 9. The probe system of claim 1,wherein the one or more probe sequences of (c) further comprise anoligonucleotide comprising sequence Y, and wherein the ligatable complexis linear.
 10. The probe system of claim 1, further comprising a pair ofPCR primers that hybridize to the one or more probes of (c).
 11. Theprobe system of claim 1, wherein the one or more oligonucleotides of (c)comprise a backbone probe of formula X-Y-Z, where Y comprises anoligonucleotide sequence, such that the ligatable complex is a circularligatable complex of formula X-A-B-Z-Y or X-B-A-Z-Y, where sequence Yjoins sequences X and Z.
 12. The probe system of claim 11, furthercomprising a rolling circle amplification primer that hybridizes to asequence in the backbone probe.
 13. The probe system of claim 11,further comprising: (A) a rolling circle amplification primer thathybridizes a sequence to the backbone probe; and (B) up to fourdistinguishably labeled detection oligonucleotides, wherein each of thedistinguishable labeled detection oligonucleotides hybridizes to a B′sequence.
 14. A method comprising: (a) hybridizing a probe system ofclaim 1 with a test genomic sample that comprises genomic fragments, toproduce ligatable complexes of formula X-A-B-Z or X-B-A-Z; (b) ligatingthe ligatable complexes to produce product DNA molecules of formulaX-A-B-Z or X-B-A-Z; and (c) counting the product DNA moleculescorresponding to each locus identifier of sequence B.
 15. The method ofclaim 14, wherein the counting is done by sequencing product DNAmolecules, or amplification products thereof, to produce sequence reads,and counting the number of sequence reads comprising each sequence of Bor complement thereof.
 16. The method of claim 14, wherein the productDNA molecules are circular, and the counting comprises amplifying theproduct DNA molecules by rolling circle amplification, and counting thenumber amplification products comprising each sequence of B orcomplement thereof.
 17. The method of claim 16, wherein the methodcomprises labelling the RCA products using distinguishably labeledprobes that hybridize to sequence B′, and the counting is done bycounting the number of RCA products for each distinguishable label. 18.The method of claim 17, further wherein the method comprises: i.depositing the RCA products on a planar support; and ii. counting thenumber of the individual labeled RCA products in an area of the support.19. The method of claim 18, wherein the support is a glass slide. 20.The method of claim 18, wherein the support is a porous transparentcapillary membrane.
 21. The method of claim 14, wherein the differentsequences of B and their complementary sequences B′ identify differentchromosomes, and the method further comprises comparing the number ofproduct DNA molecules comprising a first sequence of either B or B′ tothe number of product DNA molecules comprising a second sequence ofeither B or B′ to determine if the genomic sample has an aneuploidy. 22.The method of claim 21, wherein method comprises comparing the countingresults of (c) with the counting results obtained from one or morereference samples.
 23. The method of claim 14, wherein the test genomicsample is from a patient that is suspected or at risk of having adisease or condition, and the counting results of (c) provide anindication of whether the patient, or fetus thereof, has the disease orcondition.
 24. The method of claim 23, wherein the disease or conditionis a cancer, an infectious disease, an inflammatory disease, atransplant rejection, or a trisomy.
 25. The method of claim 14, whereinthe fragments are restriction fragments.